This is generally in the area of compositions and methods for use thereof for the treatment of oxidative tissue damage such as that resulting from ischemia or inflammation, wherein the active compounds are phenyl butylnitrone (PBN) or derivatives thereof.
Oxygenated tissue suffers damage, in many cases permanent damage, if it becomes ischemic and is then reperfused. Oxygen free radicals have been implicated in the injury. Brain appears to be uniquely susceptible to ischemia/reperfusion injury, and neurons are more susceptible than glial cells. Certain areas of the brain, for example, the hippocampus and spinal cord, are more susceptible than other regions of the brain. As a result, ischemia/reperfusion injury to brain may have a multiplicative effect simply because of the necessity for complete integrity of all regions in order to have proper functioning.
Several mechanisms may cause ischemic brain damage. For example, the mechanisms responsible for selective neuronal vulnerability may be different from those that cause glial swelling and extensive brain edema, and a third set of events may underlie such gross functional aberrations as seizures. Siesjo, Critical Care Med. 16, 954-963 (1988), has grouped the numerous observations into four possible mechanisms to explain the injury in brain. These include: (A) calcium mediated cell death, (B) excitotoxic damage (glutamate buildup), (C) free radical events and (D) acidosis. It is likely that all four basic mechanisms are either interrelated or contribute collectively to the injury observed.
It now seems likely that selective neuronal vulnerability is, at least to some extent, an excitotoxic lesion that is triggered by the release of glutamate and/or asparate from depolarized nerve endings. At the membrane and molecular levels, calcium influx via NMDA-activated channels is probably crucial, although osmolytic damage to dendritic spines and dendrites could contribute. It has never been satisfactorily explained, however, how ischemia could cause delayed neuronal death.
The mechanisms that cause laminar necrosis, glial distruction, and infarction are more speculative. The inability of the cells to regulate their volume may be caused by a molecular defect where edema is a conspicuous feature. Cells that possess coupled antiporters for Na+/H+ and Cl-/HCO.sub.3 exchange may regulate pH; at the expense of volume regulation, so that edema may be coupled to acidosis.
When ischemia is accompanied by a delayed acidosis-related damage, it seems likely that the lowering of pH causes changes in protein structure and function, which "mature" with time and ultimately cause gross membrane dysfunction. It is tempting to assume that the primary lesion is an iron-catalyzed free-radical damage to membrane components, enhanced by the drastic lowering of pHi and/or pHe.
Free radicals have been postulated to be mediators of reperfusion damage. The important production sites of such radicals as the superoxide (O.sub.2 --) and hydroxyl (OH) species are the mitochondrial respiratory chain and the sequences catalyzed by cyclooxygenase and lipoxygenase. However, radicals are also formed furing autoxidation of many compounds (e.g., catecholamines). Several ischemic events favor a spurt of free-radical formation, e.g., those causing oxidation of polyenoic free fatty acids, release and reuptake of catecholamines, and oxidation of hypoxanthine by xanthine oxidase. Although all these events occur during recirculation, when the O.sub.2 supply is restored, they represent metabolic cascades triggered by agohist-receptor interactions, energy failure, and/or calcium influx during the insult. Although free radical formation is a likely cause of ischemic damage, it has been difficult to directly demonstrate that such formation occurs and/or that it is sufficiently pronounced to overwhelm the antioxidative defense of the tissue, Curran, et al., Mol. Cell. Biol. 5, 167-172 (1985). In recent years, however, evidence has been obtained that ischemia may cause conjugated dienes and malondialdehyde to accumulate in the tissue. Nonetheless, it remains to be conclusively shown that free-radical damage to unsaturated acyl chains in phospholipids, to protein, or to nucleic acids constitutes an important part of the ischemic necrosis. At present, the evidence is relatively strong for an involvement of free-radical mechanisms in vascular injury, but not for damage affecting nerve and glial cells.
Reviews by Hall and Braughler, Free Radical Biol. Med. 6, 303-313 (1989), Kontos, Physiology of Oxygen Radicals, ed. Taylor, Matalon and Ward, pp. 207-216 (Am. Physiol. Soc., Bethesda, Md. 1986), and Ernster, Critical Care Med. 16, 947-953 (1988), document a significant amount of evidence implicating oxidative damage in head and spinal cord injury as well as in hemorrhagic stroke and ischemic stroke. Most research which strongly implicates the primary role of oxidative damage in ischemia/reperfusion injury in brain can be grouped into either of two types of studies: those which show protection by addition of agents preventing peroxidative events, or those designed to observe free radicals.
Although no drugs are currently approved for clinical use in treating tissue damage due to ischemia, several compounds have been proposed as potentially being effective. Mannitol, an osmotic agent and an oxygen scavenger, has been added to reperfusion media and may limit damage to organs for transplantation. Superoxide dismutase (SOD) has been suggested as a means for limiting in vivo oxidative damage. The most promising compounds that interfere with peroxidation generation are the lazaroids, modified prednisones, described by J. M. McCall, Acta Anesthesia Belgica, First Antwerp Int. Trauma Symp., which have been reported to be efficacious if given during or after ischemia. For example, Hall, et al, Stroke 19, 997-1002 (1988), demonstrated that the 21-aminosteroid (474006F), which is known to prevent peroxidation in brain homogenate, as described by Braughler, et al., J. Biol. Chem. 262, 10438-10440 (1987), protected gerbils against brain damage induced by three hours of unilateral carotid artery occlusion. White and Aust and co-workers, Adv. Free Radical Biol. Med. 1, 1-17 (1985), and Babbs, Resuscitation 13, 165-173 (1986), have demonstrated that iron chelators protect animals from ischemia/reperfusion injury.
With regard to direct demonstrations of oxidative events during ischemia/reperfusion injury in brain, there are pertinent observations using spin-trapping techniques, salicylate hydroxylation, protein oxidation, and nucleic acid oxidative damage. Spin-trapping has provided clear evidence implicating free radical production dunng ischemia/reperfusion injury. Imaizumi, et al, Neurological Res. 8, 214-220 (1986), showed that the spin-trap PBN when incubated with rat brain homogenate of animals which had experienced very low oxygen pressure for 5 min was able to trap an apparently lipid-type radical. Kirsch, et al, Pediatric Res. 21, 202A (1987), stated in a preliminary note that PBN had trapped free radicals in post ischemic brains from animals pretreated with the spin-trap. McCay's group have spin trapped free radicals in mouse brain, Arch. Biochem. Biophys. 244, 156-160 (1986), and shown that spin-trapped free radicals of PBN are present in blood leaving the heart of intact dogs recovering from a "stunned" myocardium, J. Clin. Invest. 82, 476-485 (1988). As described, the free radicals spin-trapped are apparently lipid-type free radicals and appear within 1 min after release of occlusion and reach peak intensity about 5 min after reperfusion starts. PBN enhanced recovery of the post ischemic function of the "stunned" heart.
In summary, while PBN has been used in a number of research studies, there has been no data to support the proposition that it could be useful in vivo, particularly with respect to treatment of tissue damage in the central nervous system. In vivo, the drug must be able to (1) cross the blood brain barrier and (2) act in a manner which reduces tissue damage during or following ischemia.
It is therefore an object of the present invention to provide composition and methods for use thereof which are useful in preventing or reversing ischemic damage in vivo., especially in the CNS, spinal cord and eyes.
It is a further object of the present invention to provide compositions and methods for use thereof which are useful in preventing or reversing free radical damage in vivo resulting from infection and inflammation.
It is another object of the present invention to provide compositions and methods for use which prevent energy depletion of cells during ischemia.
It is still another object of the present invention to provide compositions and methods for use which are useful in the treatment of a variety of disorders in addition to those resulting from ischemia, including progressive neuronal loss from disease or drug and alcohol abuse and disorders resulting from photooxidation and exposure to high pressure or enriched oxygen.